Thermo Scientific™ Pierce™ DAB Substrate Kit

The Thermo Scientific™ Pierce™ DAB Substrate Kit is a chromogenic detection reagent designed for horseradish peroxidase (HRP)-based assays in Western blotting, immunohistochemistry (IHC), and other tissue staining methods. The key component, 3,3'-diaminobenzidine (DAB), reacts with HRP in the presence of hydrogen peroxide (H₂O₂) to produce an insoluble brown precipitate at the site of HRP-conjugated antibody binding.

This ready-to-use kit simplifies HRP detection in tissue sections, membranes, and fixed cell samples, making it an essential reagent in chromogenic immunoassays. Unlike other HRP substrates, DAB precipitates are insoluble in alcohol and organic solvents, allowing compatibility with traditional counterstains and mounting media.

Key Features & Benefits

1. Chromogenic HRP Substrate:
   • Produces a brown, insoluble precipitate at HRP binding sites, allowing visual detection.
   • Works on Western blots (WB), nitrocellulose/PVDF membranes, and immunohistochemistry (IHC) applications.
2. Preformulated for Convenience:
   • Includes a 10X DAB stock solution and a Stable Peroxide Substrate Buffer for immediate use.
   • No need for complex buffer formulations or separate peroxide additions.
3. High Stability & Storage:
   • Refrigerated storage at 4°C maintains kit stability for at least one year.
   • No need for freezer storage.
4. Versatile & Compatible:
   • Suitable for Western blot (WB), immunohistochemistry (IHC), and electron microscopy (EM).
   • Compatible with PVDF and nitrocellulose membranes.
   • Insoluble in alcohol-based mounting media, ensuring long-lasting stain retention.
5. Safe & Easy to Use:
   • Preformulated stable peroxide buffer minimizes safety concerns.
   • Ready-to-use format allows for fast preparation.

Applications & Recommended Uses

The Pierce DAB Substrate Kit is widely used in HRP-based detection assays, including:

1. Western Blotting (WB):
   • Detects HRP-conjugated primary or secondary antibodies on nitrocellulose and PVDF membranes.
   • Produces brown bands corresponding to target proteins.
2. Immunohistochemistry (IHC):
   • Enables tissue staining for protein localization using HRP-conjugated antibodies.
   • Ideal for paraffin-embedded and frozen tissue sections.
3. Immunocytochemistry (ICC):
   • Facilitates HRP-based staining in fixed cell cultures.
   • Used for chromogenic labeling of cellular proteins.
4. Electron Microscopy (EM):
   • Works as a contrast agent for ultrastructural localization of proteins in EM studies.

Product Specifications

How DAB Works in HRP-Based Detection

DAB (3,3'-diaminobenzidine tetrahydrochloride) functions as a precipitating substrate in HRP-based assays. The reaction follows these steps:

1. HRP catalyzes the oxidation of DAB in the presence of hydrogen peroxide.
2. Brown precipitate forms at the site of HRP-conjugated antibody binding.
3. The insoluble brown stain remains stable even after dehydration, making it suitable for long-term storage and microscopic analysis.

Storage & Handling Instructions

• Storage Conditions: 4°C (refrigerated), no freezing required.
• Shelf Life: Stable for at least 1 year under proper storage conditions.
• Working Solution Preparation:
  • Dilute 10X DAB solution to 1X in Stable Peroxide Buffer just before use.
  • Use working solution immediately to prevent increased background staining.

Precautionary Notes:

• DAB is a suspected carcinogen; handle with proper PPE and dispose of according to safety regulations.
• Work in a fume hood and use protective gloves and eyewear when preparing DAB solutions.

Comparison with Other HRP Substrates

Key Advantage of DAB:

• Insoluble brown precipitate ensures stable staining.
• Better contrast with counterstains in IHC applications.

Related Products

For enhanced DAB staining or alternative HRP substrates, consider:

The Thermo Scientific™ Pierce™ DAB Substrate Kit (Catalog #34002) is a reliable and convenient HRP substrate kit for chromogenic detection in Western blotting, immunohistochemistry, and immunocytochemistry. Its easy-to-use format, long-term stability, and strong brown staining make it an ideal choice for visualizing HRP activity in research applications.